Our goal is to leverage our collective experience in antiviral research and development to advance towards an IND an exciting novel lead small molecule inhibitor with pangenotypic coverage against one of the most common and potentially lethal viral infections known to man--the Priority Pathogen influenza virus. Our current lead molecule, RS-8443, exploits a proprietary 4' sugar modification that makes the otherwise natural base cytosine nucleoside the most potent molecule described to date against the influenza polymerase, with an IC50 of 0.6 micromolar. Moreover, this modification renders the molecule highly specific for the influenza polymerase with no activity against other tested polymerases--thereby overcoming a key potential liability of other nucleoside analogs, and affording a lack of toxicity in tested cell lines. We now seek to develop our lead compound into a clinical stage drug by: 1) Optimizing our RS-8443 lead and identifying a back-up compound by assaying analogs with other natural bases, monophosphate prodrug moieties, or novel lung-activated prodrug moieties for their phosphorylation kinetics following delivery to standard cultured (MDCK, A549) and more relevant human primary lung target cells. We will then determine the biochemical activities (influenza polymerase, human mitochondrial RNA and DNA gamma polymerases) and antiviral efficacy of these derivatives in vitro; 2) Determining the pharmacokinetic (PK) profile and major metabolites of the optimized lead by: a) performing full rat and mouse PK studies of multiple oral and intravenous doses; and b) identifying the optimized lead's major metabolites in vitro (mouse, rat, dog, monkey and human hepatocytes) and in vivo (mouse, rat); 3) Determining key virologic and mechanistic properties of the optimized lead by: a) assessing it's in vivo antiviral efficacy (mouse model) and potential for emergence of drug resistance compared to other direct-acting antivirals (e.g. Tamiflu); b) determining the activity of the optimized lead against multiple genotypes and drug resistant mutants to amantadine and Tamiflu; and c) determining whether the optimized lead acts via chain termination vs. incorporation; 4) Nominating an IND candidate by subjecting the optimized lead, its major metabolite, and the backup compound to in vitro ADME-Tox and initial preclinical animal safety testing; 5) Completing the IND-enabling studies by conducting a scale up campaign of the IND candidate, multiple dose 28 day escalation rodent and dog studies, and preparing a successful IND application. Our multidisciplinary team--including academics and industry partners with demonstrated expertise in virology, nucleoside chemistry, PK and metabolite analysis, and successful early drug development-is ideally suited for this proposal. Successful accomplishment of our specific aims will yield an exciting novel drug capable of conferring protection against this key Priority Pathogen, including its most virulent strains that threaten millions.